Human papillomavirus type 16 E6/E7-specific cytotoxic T lymphocytes in women with cervical neoplasia

Infection with oncogenic human papillomavirus (HPV) types is associated with the development of cervical neoplasia (CIN). The E6 and E7 oncoproteins are constitutively expressed in these lesions and are therefore putative targets for the immune response against HPV. The relation between HPV 16-specific memory cytotoxic T-cell precursor (mCTLp) activity to both oncoproteins and the natural course of cervical dysplasia was analyzed in 38 patients participating in a nonintervention cohort study of women with CIN and 11 HPV 16-positive cervical carcinoma patients. In a cross-sectional study at the end of follow-up prior to biopsy, 8 of 20 patients with a persistent HPV 16 infection had specific mCTLp against at least one of the two oncoproteins. By contrast, no specific mCTLp activity was detected in 11 HPV-negative patients or in 7 patients who had cleared an HPV 16 infection at the end of follow-up. However, 5 of 11 cervical carcinoma patients showed mCTLp activity against the E7 protein only. This study demonstrates that HPV 16 oncogene-specific mCTLp are present in women with HPV 16-positive CIN prior to any intervention. Since HPV-specific mCTLp were detected predominantly in women with high-grade lesions or invasive cervical carcinoma and not in women who cleared the virus, the role of naturally occurring mCTLp in the protection against HPV-associated cervical neoplasia remains to be established.     

Deficiencies in myeloid antigen-presenting cells in women with cervical squamous intraepithelial lesions

BACKGROUND: There is little information on the function of dendritic cells in women with human papillomavirus (HPV)-related cervical squamous intraepithelial lesions (SILs). In the current study the functions of dendritic cells in the development of T-cell immunity in women with cervical SILs were assessed. METHODS: The percentage of myeloid dendritic cells (MDCs) and plasmacytoid dendritic cells (PDCs) in peripheral blood were enumerated of 44 patients with SIL (low-grade, 19; high-grade, 25), 19 patients with atypical squamous cells of undetermined significance (ASCUS), and 18 controls. The expression of costimulatory receptors was assessed and the ability of monocyte-derived dendritic cells (MDDC) to present HPV16-E6 and HPV16-E7 antigens to autologous T cells. RESULTS: Patients with either low (L)-grade or high (H)-grade SIL had significantly lower median plasma levels of interferon-gamma than did the controls (P = .038 and .031, respectively). Compared with the controls, patients with ASCUS or LSILs had significantly lower median percentages of MDCs (P = .002 and P < .001, respectively), and significantly lower median percentages of MDDCs that expressed CD86 (P < .001 and P = .003, respectively) and major histocompatability complex class-II antigen human leukocyte antigen DR (HLA-DR) (P = .012 and P < .001, respectively). T cells of patients with ASCUS or LSILs proliferated less than those of the controls in response to HPV16-E7 (P = .002 and .046, respectively). CONCLUSIONS: Low levels of peripheral blood MDCs and of MDDCs expressing CD86 and HLA-DR suggest that deficiencies in the ability of MDDC to present antigen to autologous T cells may lead to persistent infection with HPV and the development of cervical SILs in HPV-infected women.     

宫颈 TCT 异常患者行 HPV 检测在宫颈病变诊断中的临床价值

目的：探讨薄层液基细胞学检查（TCT）异常的患者行人乳头状瘤病毒（HPV）检测在宫颈癌及癌前病变诊断中的临床价值。方法：2010年3月至2012年3月在汉川市人民医院妇科门诊就诊的患者共计2389例行液基细胞学检查,结果异常的有526例患者,同时进行HPVDNA检测和阴道镜下宫颈活检,以组织病理学结果作为诊断的金标准。结果：宫颈活检组织病理学诊断结果：526例患者中宫颈炎及CINI456例,占86．69％,CINIl34例,占6．46％,CINⅢ32例,占6．08％,宫颈浸润癌4例,占0．76％。HPVDNA检测阳性者共有226例,阳性率随病变严重程度的增加而增加。CINII级及其以上病变检出率在HPVDNA检测阳性组中明显高于阴性组中,两组比较有显著性差异（P〈0．05）。结论：细胞学检查结果异常的患者,病理组织学检查结果相差较大,可以从宫颈炎症到宫颈癌。HPVDNA检测可以减少单纯应用薄层液基细胞学而造成的宫颈病变漏诊率,并对细胞学结果有进一步的分流作用。     

Tumor infiltrating lymphocytes in seminoma lesions comprise clonally expanded cytotoxic T cells

Seminoma lesions are characterized by a brisk inflammatory infiltrate containing both CD4 and CD8 T cells, which is of prognostic significance. However, whether seminoma cells express the HLA molecules required for classical T-cell recognition remains controversial. In the present study, we conducted a molecular, phenotypical and functional characterization of tumor infiltrating lymphocytes (TILs) from seminoma lesions. T-cell receptor clonotype mapping demonstrated the presence of clonally expanded T cells in the majority of the lesions. The cytotoxic capacity of TILs was indicated by expression of CD107a, which is a recently described surrogate marker for cytolytic activity. Indeed, the frequency of CD107a positive cells was substantially higher in TILs when compared to peripheral blood mononuclear cells. Moreover, fluorescence activated cell sorting of CD107a positive TILs allowed comparison of the clonotypic T-cell receptor fingerprint and demonstrated the ability of expanded clones to express this cytotoxic marker, suggesting cytotoxic activity at the tumor site. The cytotoxicity was confirmed by in situ granzyme B expression. Furthermore, by staining with multimeric HLA-peptide complexes, we could demonstrate the presence of Mage-3 specific T cells among TILs. In summary, specific and functional T-cell responses are operative in seminoma, indicating that the inflammatory infiltrate is indeed involved in the immunological control of the tumor.     

Clonal cytotoxic T cells in myeloma

Multiple myeloma (MM) is a malignant disease characterized by accumulation of morphologically recognizable plasma cells producing immunoglobulin (Ig) in the bone marrow. The occurrence of clonal T cells in MM, as defined by the presence of rearrangements in the T-cell receptor (TCR)-beta chains detected on Southern blotting, is associated with an improved prognosis. This review aims to describe the various ways in which we have demonstrated the presence of such T cell clones, and to describe the phenotype of these cells. Finally, the specificities of these clinically important CD8+ T cell populations will be discussed in the context of immunotherapy.     

The cytotoxic and mutagenic effects of alkylating agents on human lymphoid cells are caused by different DNA lesions

The Burkitt's lymphoma cell line Raji has a Mex⁺ phenotype.It is more resistant to killing by alkylating agents than asub line (Raji TK^–) which is Mex^–. A reduction inO⁶-methylguanine (O⁶MeG)-DNA methyltransferase can be broughtabout by growing Raji cells in the presence of free O⁶MeG. Thedepletion in enzyme activity is specific and reversible; removalof O⁶MeG from the medium results in the restoration of methyltransferaseactivity within 4 h. Raji cells, in which methyltransferasehas been reduced by this treatment to below detectable levels,are not sensitised to killing by N-methyl-N'-nitro-N-nitrosoguanidineor the cross-linking nitrosoureas, 1, 3-bis(2-chloroethyl)-1-nitro-soureaand 1-(2-chloroethyl)-1-nitrosourea. This implies that adductsat the 0⁶ atom of guanine in DNA are not potentially cytotoxiclesions. Secondly, it suggests that a defect other than thelack of methyltransferase is responsible for the sensitivityof Mex^– cells to killing by alkylating agents.     

The association between cytotoxic T lymphocyte-associated antigen-4 and cervical cancer

Cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) gene polymorphisms have been associated with many autoimmune diseases and malignancy susceptibility, but the relationship between CTLA-4 and cervical cancer is still controversial. Hence, a meta-analysis of the published studies for the CTLA-4 gene polymorphisms and the risk of cervical cancer was performed to evaluate the association between them. Odds ratios (ORs) and 95 % confidence intervals (CIs) for the codominant, dominant, and recessive genetic models were assessed. The fixed or random effect pooled measure was selected on the basis of the heterogeneity test among studies. The heterogeneity among studies was evaluated using the I ². Eight studies with 2,835 cases and 2,560 controls were included. In seven studies for the CTLA-4 +49A/G polymorphism, a significant association was showed between the A allele and the increased risk of cervical cancer in the codominant (OR 1.16, 95 % CI 1.05–1.29), dominant (OR 1.18, 95 % CI 1.03–1.36), and recessive (OR 1.24, 95 % CI 1.05–1.56) models. In five studies for the CTLA-4 ?318C/T polymorphism, the meta-analysis showed a significant association of the C allele with the reduced risk of cervical cancer in the codominant (OR 0.79, 95 % CI 0.66–0.94) and recessive (OR 0.76, 95 % CI 0.63–0.93) models. This meta-analysis suggested that +49A/G and ?318C/T polymorphisms of the CTLA-4 gene were significantly associated with the risk of cervical cancer. However, further studies are required to draw a solid conclusion on the relation between the CTLA-4 polymorphism and the risk of cervical cancer.     

Temporal changes of suppressor T lymphocytes and cytotoxic T lymphocytes in syngeneic murine malignant gliomas

Summary The temporal activities of suppressor T lymphocytes (Ts) and cytotoxic T lymphocytes (CTL) were investigated in a syngeneic murine malignant glioma (a methylcholanthrene-induced ependymoblastoma of C57BL/6 mouse origin, 203-glioma).After the s.c. tumor inoculation, it was suggested that both Ts and CTL were generated with target specificity against 203-glioma cells, because neither Ts nor CTL activity were seen against syngeneic EL 4 (benzpyrene-induced thymoma), allogeneic P815 (methylcholanthrene-induced mastocytoma of DBA/2 mouse origin) or YAC-1 (Moloney leukemia-induced T -cell lymphoma of A/Sn mouse origin), but only against 203-glioma. It was found that the generation of Ts preceeded that of CTL and that the turnover was faster; furthermore, Ts were generated in the thymus and spleen, while CTL were distributed in regional lymph nodes and spleen. Surface marker analysis revealed that only Lyt-1^–.2.3⁺ T-cells participated in suppressor responses in contrast to both Lyt-1^–.2.3⁺ and Lyt-1⁺.2.3⁺ T-cells participating in cytotoxic responses.The effects of adult thymectomy (ATx) on the changes of the immunized Tcell subsets were also investigated. In mice thymectomized 3 weeks previously, the Ts activity was abrogated, whereas the CTL activity increased markedly and Lyt-1⁺ .2.3⁺ T-cells were not detected. The results suggest that CTL or their precursors bearing Lyt-1⁺ .2.3⁺ phenotype and Ts bearing Lyt-1^– .2.3⁺ phenotype are short-lived lymphocytes.Accordingly, it is suggested that in tumor-bearing mice short-lived Ts are generated earliest with target specificity and, due to the reciprocal relationships between Ts and CTL activities, may have a modulating influence on CTL; furthermore, AR may alter the patterns of generation of the precursor T-cells and Ts.     

Induction of mouse anti-melanoma cytotoxic and suppressor T cells in vitro by an artificial antigen, GM3-lactone

We investigated the ability of GM3-lactone liposomes to induce anti-melanoma T cell responses in mice. GM3-lactone liposomes, like murine B16 melanoma cells, induced anti-melanoma cytotoxic T cells (CTL) and also suppressor T cells (Ts). A small dose of GM3-lactone (0.0003 micrograms/ml) was enough to generate CTL in the in vitro primary response, whereas relatively large amounts of the antigen (0.03-0.3 microgram/ml) were required for anti-melanoma Ts induction. As the epitope for anti-melanoma Ts is NeuAc but not NeuGc residue on GM3, and anti-melanoma CTL are effectively induced by either GM3(NeuAc) or GM3(NeuGc)-lactone liposomes, GM3(NeuGc)-lactone or GM3(NeuGc) liposomes have potent activity as an artificial melanoma antigen to induce anti-melanoma CTL in vitro.     

Host response to myeloma: I. Induction of cytotoxic and suppressor T cells by in vivo immunization with MOPC 104E plasmacytoma

Spleen cells from BALB/c mice immunized with mitomycin C-treated MOPC 104E plasmacytoma cells demonstrated negligible cytotoxic activity (less than 10% specific cytotoxic activity) in the 51Cr release assay. These cells exhibited increased cytotoxic activity when they were secondarily sensitized in vitro with mitomycin C-treated MOPC 104E cells. Spleen cells from normal mice showed tumor-specific cytotoxic activity when cocultured with mitomycin C-treated tumor cells at the optimal responder to stimulator ratio of 25:1. The level of cytotoxic activity obtained by in vivo primed and secondarily in vitro sensitized spleen cells did not exceed the level of activity obtained by in vitro primary sensitized cells. Significant suppression of the cytotoxic activity of in vitro primary sensitized cells was observed when cocultured with in vivo primed spleen cells during primary sensitization in vitro at a responder to suppressor cell ratio of 1:1. Suppressor cells of in vivo primed mice were removed by treatment with anti-Thy 1.2 and complement. These results suggest that spleen cells from in vivo primed mice consisted of at least two subpopulations of cells, a cytotoxic (prekiller) and suppressor T cells. Attempts to induce cytotoxic cells in vivo might have failed because of the appearance of suppressor T cells.     

新疆汉族、维吾尔族子宫颈癌及宫颈病变HPV型别分布研究

目的 了解汉族、维吾尔族宫颈癌及宫颈病变患者人乳头状瘤病毒（HPV）的感染及其基因型分布情况,并分析两族间的差异。方法 采用导流杂交基因芯片技术检测2011年11月至2012年5月在新疆维吾尔自治区人民医院住院或门诊就诊的244例宫颈癌及宫颈病变患者（汉族、维吾尔族各122例）的HPV基因型及分布。结果 244例患者中共检测出18种HPV亚型,其中HPV16型在汉族和维吾尔族中所占的比例均最高;在慢性宫颈炎和宫颈上皮内瘤变（CIN）中检测出18种HPV亚型,宫颈癌中检测出8种。随病变程度的加重,HPV亚型趋于集中,两族中趋于集中的HPV亚型为HPV16、18、52、58、53、39型,其检出率在两族患者间差异无统计学意义（P＞0.05）;汉族和维吾尔族HPV多重感染率分别为22.13％（27／122）和34.43％（42／122）,差异有统计学意义（P＜0.05）。结论 汉族和维吾尔族妇女宫颈癌及宫颈病变中HPV型别分布存在一定差异,维吾尔族HPV多重感染率高于汉族妇女,这可能是新疆维吾尔族妇女宫颈癌发生率显著高于汉族妇女的原因之一。     

Adult T-cell leukemia/lymphoma cells from blood and skin tumors express cytotoxic T lymphocyte-associated antigen-4 and Foxp3 but lack suppressor activity toward autologous CD8+ T cells

Adult T cell leukemia/lymphoma (ATL) cells share the CD4⁺CD25⁺ phenotype with regulatory T (Treg) cells. However, it is still controversial whether ATL cells are Treg cells. The aim of the present study was to investigate the Treg nature of ATL cells obtained from peripheral blood and skin tumors in terms of their phenotype and function. By flow cytometry and immunohistochemistry, the expression of the Treg-associated molecule cytotoxic T lymphocyte-associated antigen (CTLA)-4 and Foxp3 was examined in freshly isolated circulating and skin-infiltrating tumor cells from 21 ATL patients with skin eruptions. The expression of CTLA-4 on freshly isolated circulating tumor cells was elevated in two of 15 patients, and Foxp3 was expressed intracytoplasmically at high levels in three of nine patients. In five of the patients examined, skin-infiltrating tumor cells bore variously elevated CTLA-4 with high Foxp3 expression. The potentiality of ATL cells as Treg cells was further addressed by stimulating ATL cells with anti-CD3/CD28 monoclonal antibodies and monitoring CTLA-4 expression. With the stimulation, even CTLA-4-low ATL cells expressed higher levels of CTLA-4 than normal CD4⁺CD25⁺ cells. To study function, ATL cells isolated from blood and skin tumors were tested for their ability to suppress the proliferation of autologous CD8⁺ T cells stimulated with allogeneic lymphocytes. Despite the expression of CTLA-4 and Foxp3, these tumors were incapable of suppressing the proliferation of autologous CD8⁺ T cells. ATL cells are phenotypically Treg cells in at least some patients, but lack immunoregulatory functions, at least toward CD8⁺ T cells. ( Cancer Sci 2008; 99: 98–106)     

Inhibiting effects on the induction of cytotoxic T lymphocytes by dendritic cells pulsed with lysates from acute myeloid leukemia blasts

Dendritic cells (DCs) were established from 25 patients in complete remission of acute myeloid leukemia (AML). In patients during hematopoietic regeneration following chemotherapy the yield of DC was comparable to that of healthy donors. In patients, more than 2 months after chemotherapy, significantly less DC were generated. Comparison of the antigen-presenting capacity using tetanus toxoid of six AML patients and six healthy volunteers did not show significant differences. In six AML patients, lymphocytes stimulated with blast cell lysate pulsed DC were analyzed for cytotoxic activity against autologous blast cells. 8.4-35.6% of autologous blast cells were lysed by DC stimulated lymphocytes. In three of the six patients maximum lysis of target cells was achieved by unpulsed DC. Thus, it seems that in some patients blast cell lysates mediate inhibitory effects, which may explain to some extend immune escape mechanisms in AML.     

Intratumoral regulatory T cells alone or in combination with cytotoxic T cells predict prognosis of hepatocellular carcinoma after resection

Tumor-infiltrating lymphocytes (TILs) represent the host immune response to cancer. CD8(+) cytotoxic T cells (CTLs) have a central role in the elimination of tumors, while regulatory T cells (Tregs) can suppress the immune reaction. The aim of this study was to investigate the prognostic value of TILs, especially Tregs and CTLs, in hepatocellular carcinoma (HCC) patients after resection. CD3(+), CD4(+), CD8(+), and FoxP3(+) TILs were assessed by immunohistochemistry in tumor tissue from 141 randomly selected HCC patients. Prognostic effects of low- or high-density TIL subsets were evaluated by Kaplan-Meier and Cox regression analysis using the median values as cutoff. The density of intratumoral Tregs (P = 0.040) and peritumoral CTLs (P = 0.004) were an independent factor for overall survival (OS), but not for disease-free survival (DFS). The density of CD3(+) and CD4(+) TILs, and the prevalence of Tregs and CTLs were associated with neither OS nor DFS. The presence of low intratumoral Tregs with high intratumoral CTLs was a negative independent prognostic factor for OS (P = 0.001), while that of low intratumoral Tregs and low peritumoral CTLs independently correlated with improved DFS (P = 0.008). Moreover, the combined analysis of Tregs and CTLs displayed better prognostic performances than any of them alone. Additionally, higher density of intratumoral Tregs correlated with both the presence of liver cirrhosis (P = 0.025) and increased tumor size (P = 0.050). Tregs within tumor environment are promising prognostic parameters for HCC patients, and their combination with CTLs can predict prognosis more effectively.     

Analysis of human serum from women affected by cervical lesions

Cervical cancer is one of the first causes of death in Mexican women population. The plasma proteome has a wide dynamic range concentrations of different protein and their alterations reflect the physiological state of the individual's health. The aim of this study was to characterize the 2D-PAGE serum patterns from healthy women and with different levels of cervical lesions. Changes in haptoglobin, apolipoproteins, and transthyretin, when comparing the serum from healthy women and serum from patients with different levels of cervical lesion were found. The Western blot analysis showed increasing concentrations of metalloproteinases (MMP's), proteins with important biological roles in tumor development and metastasis. Protein profiles in conjunction with MS, bioinformatics, and Western blot analysis, allow us to compile information for the acquisition of results to proposed candidates biomarkers of cervical cancer among Mexican women population.     

Generation of Epstein-Barr virus antigen-specific suppressor T cells in vitro

Immunosuppression is a commonly observed phenomenon in Epstein-Barr virus (EBV)-associated disorders and malignancies. The purpose of this study was to determine whether EBV antigens could generate suppressor cell activity in vitro. Peripheral blood lymphocytes (PBL) were first treated with various concentrations of EBV antigens or culture medium for 5 days and then with mitomycin C. The cells were then washed and tested for their ability to abrogate the blastogenic response of fresh, autologous PBL to previously determined optimal concentrations of EBV antigens. It was found that excess of both EBV antigens tested (soluble antigen and virus particles) induced suppressor cells, while optimal antigen concentrations failed to do so. In addition, PBL incubated with excess of EBV antigens for 10 days, without mitomycin treatment, inhibited the response of fresh autologous lymphocytes to EBV antigens. The generated suppressor cells were found to be antigen-specific since they inhibited the response of sensitized lymphocytes to the inducing antigen only. Moreover, experiments performed using purified lymphocyte subpopulations indicated that the suppressor activity was associated with T-cell populations. Using T-cells specific monoclonal antibodies, we further determined that the inhibitory activity was due to suppressor (OKT 8+) T-lymphocytes; treatment of T-lymphocyte populations (exhibiting suppressor activity) with OKT 8 antibody and complement abrogated the inhibitory effect of these populations on the response of sensitized lymphocytes to EBV antigens. Taken together, these observations suggest that similar suppressor cells may be at least partly responsible for the immunosuppression observed in patients with an antigenic overload, particularly during persistently active virus infection or malignancy.     

Western blot analysis of antigens on melanoma cells recognized by cytotoxic T cells

Antigens recognized by cloned cytotoxic T lymphocytes (CTLs) from patients with melanoma were examined by methods based on the ability of antigens immobilized on nitrocellulose paper to stimulate proliferation of the CTLs. The proliferative response depended on the presence of histocompatible antigen-presenting cells (APCs) in the cultures in the form of either autologous lymphoid cell lines (Epstein-Barr virus-transformed B cells) or histocompatible peripheral blood lymphocytes and was maximal at 3 days. Presentation appeared to be via class II major histocompatibility complex antigens, in that monoclonal antibodies (MAbs) against the class II antigens, but not the class I antigens, on the APCs inhibited the proliferative responses. The response the CTLs appeared to be mediated by interaction with the alpha beta CD3 T-cell receptor complex, in that pretreatment of the CTL clones with MAbs against CD3 inhibited the response of these clones to the antigen extracts irrespective of the phenotypes of the clones. Extracts from several nonmelanoma cells did not stimulate CTL clones specific for melanoma. At least two different specificities were detected in extracts from autologous and allogeneic tumor cells. The specificity of proliferative responses by CD3+ CD4+ and CD3+ CD8+ CTLs appeared to be similar to their cytotoxic activity, but CTLs with the CD3+ CD16+ CD8+- phenotype had wider cytotoxic activity against target cells not stimulating proliferative responses. The antigen(s) responsible for the stimulation were shown in all instances to have a molecular mass of 48 kilodaltons. Preliminary analysis suggested that the antigen(s) have both protein and glycolipid (ganglioside) components.     

Effect of Bacillus Calmette-Guérin cell wall skeleton on the induction of the cytotoxic and suppressor T cells against syngeneic tumor in the mouse

The effect of Bacillus Calmette-Guérin cell wall skeleton (BCG-CWS), which is one of the well-established immunopotentiators, on the induction of either cytotoxic or suppressor T cells against syngeneic tumor S1509a was investigated by an in vitro assay system. BCG-CWS showed an apparent augmenting activity in the induction of cytotoxic T cells against a syngeneic tumor when it was administered subcutaneously into mice with mitomycin C (MMC)-treated tumor cells in a form of oil-in-water emulsion. In addition, cytotoxic T cell activity was dominantly generated in regional lymph nodes. Furthermore, the immunized animals developed stronger resistance against the second inoculum of the same viable tumor cells. However, BCG-CWS had no effect on the induction of suppressor T cells in tumor-bearing host (TBH), and could not induce cytotoxic T cells against the homologous tumor in TBH in which suppressor T cells had been rapidly activated. The growth of tumor was neither suppressor nor enhanced, even if an oil-attached BCG-CWS was administered in TBH.